肝癌细胞HepG2外泌体对其自身生长行为的影响及其机制研究

Effects and mechanism of hepatic cancer cell HepG2 exosomes on their growth behavior

目的探讨肝癌细胞HepG2外泌体对细胞增殖、迁移、周期和凋亡的影响及其机制。方法培养肝癌细胞HepG2并用差速离心法提取其外泌体,采用粒径分析、透射电镜、蛋白质印迹法(Western blot)等方法对其进行鉴定。将外泌体与肝癌细胞HepG2共培养,采用细胞划痕实验比较其对细胞增殖能力的影响;采用Transwell实验比较其对细胞迁移能力的影响;采用流式细胞仪分析其细胞周期变化;采用实时荧光定量PCR检测实验组和对照组的细胞周期蛋白(Cyclin D)、细胞周期蛋白依赖性激酶调节亚基1(CKS1)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关x蛋白(Bax)的相对表达水平。结果提取后的外泌体经过Western blot实验,CD63和CD9均有条带出现,颗粒大小在30~100 nm,电镜形态正常。与对照组比较,实验组的细胞增殖率较高,肿瘤迁移能力提升;细胞G1期缩短,G2期延长;Cyclin D、CKS1和Bax相对表达水平均上升,Bcl-2相对表达水平降低。结论肝癌细胞HepG2外泌体通过调控Cyclin D、CKS1基因缩短其细胞周期,调控Bax、Bcl-2基因抑制细胞凋亡,从而对其增殖和迁移能力起促进作用。

Objective To investigate the effects of exosomes from hepatic cancer cell HepG2 on cell proliferation,migration,cell cycle and apoptosis and try to explore its mechanism.Methods The exosomes of hepatic cancer cell HepG2 were extracted by differential centrifugation,and identified with nanoparticle size analyzer,electromicroscope and Western blot.The effects of hepatic cancer cell HepG2 exosomes on their proliferation were evaluated with cell scratch test;the effects of HepG2 cells exosomes on their migration ability were estimatied with transwell experiment;the cell cycle changes were recorded by flow cytometry;the relative expression levels of cell cycle regulatory genes Cyclin D,cycline dependent kinase regulatory sulaunit 1(CKS1)and B-cell lymphoma-2(Bcl-2),Bcl-2 related x protein(Bax)were detected with real-time fluorescence quantitative PCR.Results The extracted exosomes were subjected to Western blot experiments.Both CD63 and CD9 had bands,the particle size was 30-100 nm,and the electron microscope morphology was normal.Compared with the control group,the cell proliferation rate of the experimental group was higher than that of the control group,and the tumor migration ability was significantly improved;the cell G1 phase was shortened,and the G2 phase was prolonged;the relative expression levels of Cyclin D,CKS1 and Bax increased,the relative expression level of Bcl-2 decreased.Conclusion The exosomes of HepG2 can shorten the cell cycle by regulating Cyclin D and Cks1 genes,and inhibit apoptosis by regulating Bax and Bcl-2 genes,thus promoting the proliferation and migration of HepG2 cells.